Title - Functional elucidation of the sequence-encoded regulatory activity of enhancers in vivo in the brain P.I. ? Alex Nord, PhD Research Summary: Once considered junk, non-coding regions of the genome have emerged as central components of evolution, development, and disease. The most common non-coding regulatory elements in the human genome are enhancers, which ensure expression of target genes at the right time in the right cells by controlling their activation. While recent efforts have made headway annotating enhancers in the genome, characterization of their sequence-encoded function remains a major challenge. This represents a significant barrier in understanding the important role of enhancers in complex biological processes, as well as in interpreting the effect of enhancer sequence variation on human evolution and disease. Rapid adoption of whole genome sequencing promises to amplify this issue, since considerably more potential causal mutations will be identified than can be functionally classified using current approaches. As such, there is a critical need for effective mechanisms to functionally characterize regulatory DNA at the sequence level to reveal the role of non-coding elements in human development. Massively parallel reporter assays provide a potential solution toward dissecting sequence-encoded enhancer activity, enabling quantitative measurement of hundreds to thousands of individual candidate enhancer sequences in a single experiment. Applying this approach in vivo could illuminate the normal versus pathogenic functions of enhancers in the assemblage of cells comprising mammalian tissues. It also could reveal their role mediating gene-by-environment interactions in complex conditions, such as learning and stress. In preliminary experiments, we adapted a high-throughput reporter assay to characterize enhancer function in mouse frontal cortex, and we applied single cell transcriptomics to define enhancer function in vivo in the developing mouse brain. These novel approaches open exciting long- term research avenues, enabling testing of enhancer sequence variation and cell-specific function in vivo at a scale and resolution not previously possible. We will couple our powerful functional assay with single cell genomics to address significant questions regarding gene regulatory wiring in the brain. We propose to: 1) Extend our function-based methods for the study of enhancer activity in vivo in mouse brain, and 2) Apply these methods to characterize the function of enhancers we previously mapped that control stage-specific gene expression associated with neuroplasticity, and to test the consequences on in vivo enhancer function of non-coding variation linked to brain evolution and pathology. These scientific questions build on our previous work on enhancers in the brain, using our new methods to transform the scope at which we can address key gaps in the understanding of enhancer function and fundamental questions of gene regulation in the brain. This work will have long-term impact, generating powerful new methods for studying enhancer function and revealing how gene regulation in the brain is encoded at the DNA sequence level, with an eye toward illuminating the non-coding genetic circuitry underlying human evolution, health and disease.